Biochemical Studies During Sequential stages of Root and Shoot …abap.co.in/sites/default/files/Paper-9_21.pdf · 2015-11-02 · organized structures in tissue culture in under the

Current Trends in Biotechnology and PharmacyVol. 9 (4) 380-388 October 2015, ISSN 0973-8916 (Print), 2230-7303 (Online)

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AbstractPresent investigation was carried out on

Cardiospermum halicacabum L. (Ballon Vine) -an important medicinal plant. Leaf explants werefound to be the best for induction and growth ofcallus. Depending on fresh and dry weight ofcallus, high growth value callus was obtainedon MS medium supplemented with 2, 4-D (1.0mg/l) in combination with Kn (1.0 mg/l). Thiscallus was selected for further studies of root andshoot organogenesis as well as biochemicalstudies. Regeneration of roots (15 days) fromcallus was observed on MS medium + BAP (0.5mg/l) + NAA (0.3 mg/l) while shoots (16 days) onMS medium + BAP (0.5 mg/l) + NAA (0.1 mg/l).Starch content and reducing sugars were high incontrol or undifferentiated callus, which furtherincreases significantly in root and shootdifferentiating cultures. Contents of total solublesugars, total soluble proteins and total phenolswere lower in the control callus which increasesin the root and shoot differentiating cultures. Theactivities of enzymes i.e. a-amylase, acidprotease and peroxidase increased up toappearance of green patches (8th day) and werereached at peak on 12th day, the day that coincidewith the visual appearance of roots and shoot.Conversely, the acid invertase activity decreasestill the appearance of roots and shoots.

Key words: Ballon vine, callus, metabolites,enzymes, differentiation.

Abbreviations: MS, Murashige and Skoog; 2,4-D, 2, 4-dichlorophenoxy acetic acid; IAA,indole acetic acid; IBA, á-indole 3-butyric acid;NAA, á-naphthalene acetic acid, BAP, 6-benzylamino purine; Kn, kinetin.

IntroductionMedicinal plants are the most exclusive

source of life saving drugs for the majority ofthe world’s population and are important forsocio-economic uplift of the human being. WorldHealth Organization (WHO) has enlisted over21,000 plants which have medicinal value.More than 2000 plant species are used intraditional medicines as evident from byCharak Samhita and Sushruta Samhita and159 pharmaceutical companies and 3.5 billionpeople rely on these traditional medicines (5).Cardiospermum halicacabum L. (Ballon Vine) isan important medicinal plant belonging to familySapindaceae. It is an annual or perennial climber.The medicinal properties of this plant are due to(b-sitosterol, D-glucoside, saponins andquebrachitol, stigmasterol, proanthocynidia andapigenin) present in the leaves. Knowledge onthe control of differentiation has hardly grownsince the demonstration that differentiation oforganized structures in tissue culture in underthe influence of growth regulators like cytokininsor a combination of cytokinin and auxin alongwith other components of the culture medium

Biochemical Studies During Sequential stages of Rootand Shoot Differentiation in Callus cultures of

Cardiospermum halicacabum L.

Ashwani Kumar1, 2*, S.C. Goyal1, Pooja1, Charu Lata2 and Jagdish Parshad3

1 Department of Botany & Plant Physiology, CCS HAU, Hisar – 125004, Haryana, India2ICAR-Central Soil Salinity Research Institute, Karnal – 132001, Haryana, India

2Department of Microbiology, CCS HAU, Hisar – 125004, Haryana, India*For Correspondence - [email protected]

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(23). Although remarkable progress has beenmade in the area of gene transfer technology,little is known as to how plant cells differentiatein cultures or about molecular mechanism of invitro differentiation (9). This unique property alsooffers an opportunity to investigate cellular andmolecular basis of differentiation. Little is knownabout the intervening biochemical eventsoccurring in the cultured cells undergoingorganogenesis (plant regeneration), thereforeelucidation of biochemical changes accompany-ing differentiation, will be decipher the underlyingmechanism (22). There are only few reports onbiochemical studies related to in vitro rootorganogenesis from callus in medicinal plants (8,13, 16, 22, 25) in different plants. Therefore,analysis of various cellular metabolites andenzyme activities provide a reasonable andpromising approach towards an understandingof the biochemical basis of the developmentalpathway.

Materials and MethodsPlant material and growth conditions: Theplant material for this study was procured frommature plants grown in the Botanical Garden ofthe Department of Botany and Plant Physiology,CCS HAU, Hisar. The leaves obtained from fieldgrown plants of C. halicacabum were surfacesterilized with teepol solution for 10 min withvigorous stirring followed by washing in runningtap water. These were then surface sterilized with70% ethanol (1 min) followed by 0.1% mercuricchloride (3 min) and finally washed with steriledistilled water (4-5 times). The sterilized leaveswere cut into small pieces (1 cm) and immediatelyinoculated, aseptically in the flasks containing MS(18) basal medium supplemented with 1.0 mg/l2, 4-D and 1.0 mg/l Kn for callus initiation. Thecultures were kept under photoperiod (2000 lux)of 16 h light at 26 ± 2°C. The high growth callusformed on 2, 4-D and Kn was sub-cultured onMS medium supplemented with BAP (0.5 mg/l)and NAA (0.3 mg/l) for root regeneration and MSmedium supplemented with BAP (0.5 mg/l) andNAA (0.1 mg/l) for shoot regeneration. Variousmetabolites (starch, total soluble sugars,

reducing sugars, total soluble proteins and totalphenols) and enzymes (á-amylase, acidinvertase, acid protease and peroxidase)activities were assayed during the sequentialstages of roots and shoots differentiation fromcallus in Cardiospermum halicacabum asfollowing:

1. Control- undifferentiated callus before kepton differentiating medium.

2. Part of the same callus (high growth value)used for control was kept on rootdifferentiation medium [MS medium + BAP(0.5 mg/l) + NAA (0.3 mg/l)] and samplingswere done at 4th, 8th, 12th and 16th dayintervals respectively.

3. Part of the same callus (high growth value)used for control was kept on shootdifferentiation medium [MS medium + BAP(0.5 mg/l) + NAA (0.1 mg/l)] and samplingswere done at 4th, 8th, 12th and 16th dayintervals respectively

Extraction of metabolites: Extraction ofmetabolites was done by the modified methodof Barnett and Naylor (2). One hundred mg ofdry callus was homogenized in 80% ethanol (v/v) and centrifuged for 10 min at 10,000 g. Theextraction procedure was repeated three timeswith the residue. The supernatants were pooledand the final volume was made to 5 ml withethanol and used for estimation of total solublesugars, reducing sugars and total phenols byusing methods of Yemm and Willis (26), Hondaet al. (11) and Amorim et al. (1), respectively. Thepellet was hydrolyzed with 4 ml of chilled 0.2 NHClO

4 at 4°C for 24 h. The hydrolysate was

centrifuged at 5000 g for 15 min and thesupernatant was used for starch estimation bythe method of Hassid and Neufeld (10). Totalsoluble protein in the callus was extracted in 0.1M Tris-HCl (pH 7.5) by using pre-chilled pestleand mortar. The homogenate obtained wascentrifuged at 10,000 g for 15 min at 4°C andtotal soluble proteins in the supernatant wereestimated using the method of Bradford (4).

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Enzymes activity analysis : One gram of freshcallus tissue was hand homogenised in pre-chilled pestle and mortar using 0.1 M Tris-HCl(pH 7.5) buffer containing 0.25 mM EDTA, 2.5mM Cystein-HCl and 0.1% PVP. Thehomogenate was centrifuged at 10,000 g for 15min at 4°C. The supernatant was used forestimating the enzyme activity of variousenzymes viz. á-amylase, acid-invertase, acid-protease and peroxidase using the methodsfollowed by Shuster and Gifford (21), Summer(24), Beevers (3) and Seevers et al (20),respectively. Total soluble proteins in the enzymeextract were determined as per the method ofBradford (4).

Statistical analysis: All experiments wererepeated at least twice, using 8-10 replicates(flasks) each containing three explants. The datawere analyzed statistically using completelyrandomized design and the significance wastested at 5% level of critical difference.

Results and DiscussionCallus induction and root differentiation:Among different explants (leaf, node andinternode) used for callus induction and callusgrowth, the leaf explants was found to be the

Fig. 1. Depicting high growth value callus (interms of fresh and dry weight) from leaf explantson MS medium supplemented with 1.0 mg/l 2,4-D and 1.0 mg/l Kn in C. halicacabum.

Fig. 3. Depicting formation of shoots on MSmedium supplemented with 0.5 mg/l BAP and0.1mg/l NAA from leaf derived callus in C.halicacabum.

Fig. 2. Depicting formation of roots on MSmedium supplemented with 0.5 mg/l BAP and0.3 mg/l NAA from leaf derived callus in C.halicacabum.

best for induction and growth of callus. Callusinduction was observed from the cut ends of theleaves on MS medium supplemented withvarious concentrations of 2, 4-D and Kn within15 days of inoculation. But on the basis of freshand dry weight of callus, MS mediumsupplemented with 1.0 mg/l 2, 4-D and 1.0 mg/l Kn (Fig. 1; Table 1) was found to be the best for



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Gloriosa (12), Coffea arabica (14), Festucaarundinacea (19) where 2, 4-D in combinationwith Kn was also used for callus induction. The 4week-old yellowish white fragile callus sub-cultured on MS medium along with BAP (0.5 mg/l) + NAA (0.3 mg/l) showed visible roots formation(Figure 2) within 12-16 days and by decreasingthe concentration of NAA i.e. 0.1 mg/l showedshoot formation (Fig.3; Table 2).

good callus induction as well as growth with 100%callusing within 23 days of inoculation.

Any increase or decrease in concentrationof both the growth regulator resulted in thereduction of callus induction and subsequentgrowth (Table 1). The explants therefore, requirean optimum concentration of growth regulatorsfor their differentiation into unorganized callus.In many other plants like Medicago arborea (16),

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Cellular metabolitesRoot differentiation: starch content which washigher in control, increased from 0 to 8th day, afterthat the content decreased continuously till 16th

day while total soluble sugars showed sharpincrease up to 8th day then declined gradually upto root formation i.e. on 16th day (Fig. 4A). Priorto inoculation on rooting medium, the reducingsugars were more in the callus but a sharpdecrease was observed during root formation.After the appearance of roots on 12th day, a littleincrease in the content of reducing sugars wasobserved in differentiating calli (Fig. 4A). Thedecrease in starch content could be due todecrease in the activity of synthesizing enzymesor increase in hydrolyzing enzymes. The declinein total soluble sugar content was associated withutilization of sugars for growth and differentiationprocess. Similar trend was reported in Zamiafurfuracea (8) and Chlorophytum borivilianum(22).

Whereas, the total soluble proteins in rootforming callus were increased up to 12th day ascompared to control (Fig. 4B). Maximum proteincontent was found during roots initiation but afterroots formation total soluble proteins startdecreasing. During differentiation the cells arequantitatively changing their activities, new

proteins have to be synthesized, thus proteincontent in callus forming roots is high. Similarobservations were also reported by Mohapatraand Rath (17). The phenols were decreasedduring roots differentiation. On 12th day, rootsdifferentiating calli had very less phenolic contentas compared to undifferentiated callus (Fig. 4B).However, a little increase was observed on 16th

day i.e. after roots formation. Phenols contentdecreased during differentiation could be due tothat phenols participate in formation of cross-linking of cell wall constituents, which is catalyzedby peroxidase (15).

Shoot differentiation: starch content showed azig-zag trend i.e. increases from 0 to 4th day, thendecreased on 8th and 12th day and then a littleincrease was found with the appearance ofshoots on 16th day. Total soluble sugars showedsharp increase up to 4th day and declinedgradually up to shoot formation i.e. on 16th day. Asteady increase in reducing sugar content of calliwas observed before shoot initiation i.e. upto 8th

day but sugar content decreased during shootformation (Fig. 5A).

Similarly, the total soluble proteins in shootforming callus remained high throughout the

Fig. 4. Changes in the levels of starch, total soluble sugars and reducing sugars, A; total phenolsand total soluble proteins, B; in C. halicacabum callus prior to inoculation on root forming medium(0 day) and after 4, 8, 12 and 16 day of inoculation (vertical lines indicate ± SE).



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period of study as compared to control but themaximum content was found before greenpatches formation i.e. up to 8th day. The totalphenolic also showed a zig-zag trend. However,shoot differentiation was accompanied by a dropin the phenolic content (Fig. 5B).

Enzyme activityRoot differentiation: The a-amylase activity waslow in undifferentiated calli. After the transfer ofcallus on rooting medium, the a-amylase activity

increased up to 4th day and then a little decreasewas observed up to 8th day (Fig. 6A). After that amarked increase was observed up to rootformation. Whereas the undifferentiated callushad more acid invertase enzyme activity ascompared to differentiated ones. The enzymeactivity decreased up to 4th day fromundifferentiated callus. After that the activityincreased up to 12th day and then decreased upto 16th day i.e. root forming callus (Fig. 6A).

Fig. 5. Changes in the levels of starch, total soluble sugars and reducing sugars, A; total phenolsand total soluble proteins, B; in C. halicacabum callus prior to inoculation on shoot forming medium(0 day) and after 4, 8, 12 and 16 day of inoculation (vertical lines indicate ± SE).

Fig. 6. Changes in the levels of á-amylase and acid invertase, A; acid peroxidase and acid proteaseB; enzymes activity in C. halicacabum callus prior to inoculation on root forming medium (0 day)and after 4, 8, 12 and 16 day of inoculation (vertical lines indicate ± SE).

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The decrease in carbohydrate and thestarch content in differentiating callus ascompared to undifferentiated callus and higherá-amylase activity during differentiation seem dueto the mobilization of carbohydrate reserves.Similar observation was reported by Singh et al.(22). Since the acid invertase is concerned withsucrose uptake and removal of sugars from thevacuole, the observed decrease in reducingsugars content and acid-invertase activity duringdifferentiation may have a direct correlation withdifferentiation as suggested by Cuadrado et al(6). Acid-protease activity was least in control andit increased continuously up to 8th day afterinoculation. Though a little decrease in contentwas noticed during root formation i.e. on 12th daywas observed. Whereas the activity of acid-peroxidase was more in root differentiating callusthan that of control callus. The peak activities ofthis enzyme were recorded on 8th day i.e. beforeroots formation (Fig. 6B). Higher proteolyticactivity indicates high rates of degradation of pre-existing storage and other proteins requiredduring differentiation as reported in Cuminumcyminum (7) and Chlorophytum borivilianum (22).

Shoot differentiation: The activity of á-amylaseshowed little increase up to 8th day, with sharp

increase (more than 10 times) up to formation ofgreen patches (12th day) followed by a sharpdecrease in the activity on 16th day at shootsformation (Figure 7A). The acid invertase activitywas more in callus prior to inoculation fordifferentiation and showed a continuousdecrease in shoot differentiating calli (Fig. 7A)and showed a positive correlation with thereducing sugar content.

Acid protease activity was found to behigher under shoot forming conditions. Peakactivity was observed on 8th day and was about2 times higher than that in the undifferentiatedcallus (Fig. 7B). Total proteins were positivelycorrelated with protease activity. The activity ofperoxidase was more in callus than that of shootsdifferentiating calli. The peak activity of theenzyme was recorded on 8th day, associated withrapid differentiation (Fig. 7B).

SummaryThe results from present study concluded

that leaf explants gave best response for callusinduction. Indirect regeneration was achievedthrough high growth value callus formed on MSmedium supplemented with 2, 4-D (1.0 mg/l) +Kn (1.0 mg/l). MS medium supplemented with

Fig. 7. Changes in the levels of á-amylase and acid invertase, A; acid peroxidase and acid proteaseB; enzymes activity in C. halicacabum callus prior to inoculation on shoot forming medium (0 day)and after 4, 8, 12 and 16 day of inoculation (vertical lines indicate ± SE).



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BAP (0.5 mg/l) + NAA (0.1 mg/l) showed shootregeneration while the increase in NAAconcentration in the medium i.e. NAA (0.3 mg/l)showed root regeneration from the callus. Visiblemanifestation of cell differentiation includesgreening of callus, variation in cell wallthickness but differentiation in such tissuesinvolves differences in basic metabolicpathways i .e. ut i l izat ion/formation ofmetabolites and changes in the enzymeactivities. The results showed that decrease inmetabolites like starch, reducing sugars, totalssoluble sugar and total phenols while increasein total soluble proteins was found during rootand shoot differentiation. Activities of enzymesviz. á-amylase acid invertase and peroxidasedecreased while acid protease activity increasedduring root and shoot differentiation.

AcknowledgementsThe authors are thankful to Prof. and Head

Department of Botany and Plant Physiology, forhis cooperation and help during the course ofinvestigation. The CCS, Haryana AgriculturalUniversity, Hisar-125004 (Haryana) India, is alsoacknowledged for financial assistance, given tosenior author in the form of merit scholars.

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Biochemical Studies During Sequential stages of Root and Shoot …abap.co.in/sites/default/files/Paper-9_21.pdf · 2015-11-02 · organized structures in tissue culture in under the